Electrochemiluminescent measurement techniques derive from electrochemistry and chemiluminescent detection techniques. Electrochemistry (EC) deals generally with the relation of electricity to chemical changes and with the interconversion of chemical and electrical energy. Chemiluminescence (CL) based assay or detection techniques include, for example, binding assay techniques which generally comprise forming a mixture of a sample containing an unknown amount of an analyte of interest to be determined with a known amount of a reactant which is conjugated with a chemiluminescent label. The mixture is incubated to allow the labeled reactant to bind to the analyte. After incubation, the mixture is separated into two fractions: a bound and an unbound fraction. The bound fraction is labeled reactant bound to analyte and the unbound fraction is the remaining unbound reactant. The CL measurement is then taken. One or both fractions are chemically caused to luminesce, for example by the addition of an oxidant to the fractions. The bound and unbound fractions of the labeled reactant will emit different amounts of light. The measured level of chemiluminescence at the specific wavelength is indicative of the amount of the bound and/or unbound fraction, respectively, and from such measurements one skilled in the art can determine the amount of analyte in the sample.
Electrochemiluminescent (ECL) detection techniques provide a sensitive and controllable measurement of the presence and amount of an analyte of interest. In ECL techniques, the incubated sample is exposed to a voltammetric working electrode, that is, an electrode to which a voltage is applied and from which a current for a redox reaction may be passed. The ECL mixture does not react with the chemical environment alone, as does the CL mixture, nor with an electric field alone as in EC, but rather, electrochemiluminescence is triggered by a voltage impressed on the working electrode at a particular time and in a particular manner to controllably cause the ECL moiety to emit light at the electrochemiluminescent wavelength of interest. The measurement of interest is not the current at the electrode, as in EC, but rather is the intensity of the light. The ECL operating conditions should be controlled to enhance the accuracy and precision of this measurement.
The key to this control, however, is recognizing which operating conditions have an effect on the ECL measurements, how these effects appear and how the ECL measurement process may be controlled so as to provide measurement results which are reproducible within very strict limits. The chemistry involved in the ECL compounds, the analytes of interest and/or the buffer solutions in which they appear is highly complex. The ECL compounds must react with a precursor component so as to emit light. While the general nature of the chemical changes and reactions which occur during the ECL measurement process is currently believed to be known, the specific nature is not known with sufficient accuracy to permit the theoretical prediction of all factors which contribute to each measurement and to what extent they contribute and/or combine.
Nevertheless, it would be highly advantageous to provide an apparatus whose operation is controllable so that at least the initial conditions for each measurement are exactly and precisely obtained. One aspect of the control of the initial conditions relates to the surface condition of the working electrode which triggers the ECL reaction. EC techniques may include cleaning and conditioning the surface of the working electrode so as to improve its measurement of current.
It is the discovery of the present inventors that techniques which improve the measurement of current in conventional EC techniques are not necessarily desirable or useful for ECL techniques. The initial conditions for ECL measurements must meet different criteria. The analysis of results for cleaning by conventional EC techniques are based on the current response, where ECL techniques use the criteria of light intensity to evaluate the results.
It is also a discovery of the present inventors that the precision and detection limit of the ECL measurements of light are very sensitive to the condition and the redox (reduction/oxidation) state of the working electrode surface and in what manner that redox state is achieved and maintained. Conventional procedures for cleaning and/or conditioning solid voltammetric working electrodes have involved, for example, flaming, polishing, roughening the electrodes, usually followed by an electrochemical pretreatment. These procedures, disadvantageously, have frequently required the working electrode to be removed from the cell and/or manually cleaned.
It is believed that non-reproducible results during ECL measurements derive at least in part from the non-reproducible changing surface redox state of the working electrode due to variable conditions during and following the conventional cleaning/conditioning procedures. Additionally, when ECL techniques are performed on biological sample matrices, for example, serum or plasma based samples, some of the biological molecules may react with the working electrode during a measurement and cause electrode fouling.
Another aspect of the control of the ECL measurements is the relationship between the nature of the analyte of interest, the manner in which the sample is introduced into the ECL measurement cell and the use and operation of the working electrode therein. Previously ECL measurements of samples with complex biological or biochemical components, were performed only with the sample at rest in a "beaker" or batch system. In order to provide more rapid assay methods, continuous or flow systems which need not be disassembled for cleaning are needed.